Integrins are heterodimeric receptors which mediate vital cell-cell and cell-matrix interactions such as those taking place during morphogenesis, growth and differentiation, angiogenesis, hemostasis, inflammation and gene expression. Integrins bind in a divalent cation dependent manner to diverse ligands including certain plasma proteins, and extracellular matrix and membrane proteins. Despite the diversity of integrin ligands, an acidic residue is an essential feature of their integrin binding motif. In contrast to other receptors where binding to ligand is often intrinsic, integrin binding to many physiologic ligands is not constitutive but rather inducible, and is associated with conformational changes in integrins that extend to their ligand binding regions. The activation-dependent interaction of integrins with ligands plays a crucial role in regulating cell-cell and cell-matrix adhesion and in avoiding serious damage to host tissues. The overall goal of this proposal is to elucidate the structural basis of integrin-ligand interactions and to define in molecular terms the nature of integrin activation. During the previous funding period, we have identified a major ligand binding site within a conserved 200 amino acid domain (A-or I-domain) in the extracellular region of the alpha (CD11b) subunit of integrin CD11b/CD18. This domain contains a novel metal binding site which is required for the interaction of the domain as well as the holoreceptor with several ligands. We also solved the 3-dimensional crystal structure of this domain. A glutamate residue from another molecule completes the coordination of the solvent-exposed metal ion in the structure. We proposed that this arrangement may be a mimic of the interaction of an integrin with ligands, and may explain the indispensable role of acidic residues in integrin binding motifs. Hydropathy plot analysis of a highly conserved metal- and ligand binding region in all integrin beta subunits revealed a striking similarity to the alpha subunit A-domain, and we suggested that an A-like domain is also present in all integrin beta subunits. We also found that the A- domain exists in two conformations, and we proposed based on indirect structural comparisons with G proteins that the two conformations may represent active and inactive states of the domain. The presence of binding sites for several ligands in the A-domain also suggested the feasibility of targeting this domain to generate novel antagonists. In this application, we therefore propose three specific aims: 1) to derive the 3-D structure of the CD11b A-domain in complex with a ligand- derived peptide and evaluate the functional status of the two A-domain conformations. 2) to solve the 3-D structure of the A-like domain in integrin beta subunits. 3) To generate specific antagonists of the CD11b/CD18 receptor using a genetic approach. Biochemical, structural-, cell- and molecular biology techniques will be used. The derived information will be invaluable in developing a structure-based understanding of integrin ligand binding and activation. Given the potential role of CD11b/CD18 in inflammation and injury, derivation of specific antagonists of this receptor could have therapeutic benefits.